PHAII is an autosomal dominant disease of hyperkalemia and hypertension. PHAII patients also demonstrate acidosis, hypercalciuria, reduced bone density, and enhanced sensitivity to thiazide diuretics (2
). Thus, the PHAII phenotype is the opposite of the Gitelman syndrome phenotype. Gitelman syndrome is a disease in which NCC mutations (12
) disrupt NCC function (6
). Recently, mutations in WNK kinases were shown to cause PHAII (4
), suggesting that these kinases regulate Na and Cl transport. The current results indicate that WNK kinases regulate NCC activity, defining a novel Na regulatory pathway that may play a central role in controlling sodium and potassium homeostasis in humans.
The thiazide-sensitive Na-Cl cotransporter is expressed at the apical membrane of epithelial cells lining the DCT (13
). This transport protein is a member of the cation-chloride cotransporter gene family and reabsorbs 3–7% of filtered NaCl (13
). It is the target of thiazide diuretics, drugs that remain the first-line treatment for essential hypertension (14
). Gitelman syndrome, an autosomal recessive disease of hypokalemic metabolic alkalosis with hypokalemia (12
), is caused by NCC mutations, many of which generate misfolded proteins that do not reach the plasma membrane (6
). We have previously investigated NCC (SLC12A3
) as a candidate gene for PHAII and did not find linkage (3
). The present results implicate NCC in the pathogenesis of PHAII but suggest that its involvement is secondary to regulation by WNK kinases.
When expressed in oocytes with NCC, WNK4 suppressed NCC-mediated Na uptake by more than 85% compared with oocytes injected with NCC alone. This indicates that WNK4 is a potent regulator of NCC activity. Coupled with the observation that WNK4 is expressed along the distal nephron (4
), this suggests that WNK4 kinase participates importantly in regulating salt excretion in vivo. The results further suggest that the effect of WNK4 to suppress NCC activity is mediated largely by a reduction in NCC delivery to the plasma membrane. While NCC mutations that cause Gitelman syndrome also reduce plasma membrane NCC delivery, coexpression of WNK4 with NCC did not disrupt processing of nascent NCC. This suggests that WNK4 does not impair NCC folding, but rather interferes with a regulated mechanism for membrane trafficking.
WNK4 has been shown, in preliminary results, to possess kinase activity (16
). Thus, one mechanism by which it could inhibit NCC is by phosphorylating the transporter. Gamba and colleagues (17
) showed that NCC activity is modestly inhibited by PKC. Yet the effects of PKC are quite modest in comparison with the dramatic effects of WNK4. This suggests that, if direct NCC phosphorylation is responsible for the WNK4 effects, the phosphorylation site may be different from that targeted by PKC. Further experiments will be necessary to determine whether the kinase activity of WNK4 is necessary for its effects on NCC function, but WNK kinases possess non-kinase domains, including the coiled coil domains, that may participate in kinase-independent mechanisms (5
). Furthermore, a kidney-specific isoform of WNK1 was recently described that lacks the kinase domain due to alternative splicing (18
). The role of this kinase-deficient WNK kinase, versus the kinase-intact WNK kinase used in the current experiments, remains to be determined.
Based on the effects of WNK4 to inhibit NCC activity, we tested whether the mutant WNK4s retain NCC-inhibiting activity. When expressed in Xenopus
oocytes, two of three PHAII-causing mutations retain their ability to inhibit NCC activity. A third, however, Q562E, is significantly less potent than wild-type WNK4 at inhibiting NCC activity. A loss of NCC-inhibiting activity would increase distal Na and Cl reabsorption, leading to the PHA phenotype, yet most loss-of-function mutations lead to autosomal recessive, not dominant, disorders. One explanation for an autosomal dominant loss-of-function mutation would be a dominant negative effect. We did not detect a dominant negative effect of the mutant WNK4 proteins in the present experiments (data not shown), although some autosomal dominant transport disorders are not successfully recreated using the oocyte expression system (19
). It should also be emphasized that detecting an effect of mutations using the oocyte expression system may not recapitulate a defect that occurs in vivo. Thus, additional experiments will be necessary to identify the mechanism by which WNK mutations cause PHAII.
WNK4 mutations could also lead to the PHAII phenotype by acting on NCC in a tubule segment–specific manner. The mammalian DCT of most species (including human) contains two distinct segments (20
). All transepithelial Na reabsorption by DCT1 traverses an electroneutral pathway with Cl. Along DCT2, however, electroneutral Na reabsorption by the NCC operates in parallel with electrogenic Na reabsorption via the epithelial Na channel, ENaC (20
). Less NCC is usually expressed along DCT2 than along DCT1, corresponding to lower rates of electroneutral Na reabsorption along the “late” distal tubule (22
). WNK4 mutations might relieve constitutive suppression of NCC activity along DCT2, leading to increased Na and Cl transport along this segment. Wilson and colleagues (4
) suggested, based on the effect of Na salts to increase K excretion and on the paracellular localization of WNK4 expression, that WNK4 may affect paracellular chloride permeability. The present results do not exclude a role for WNK4 in mediating or regulating paracellular permeability in addition to its effect on NCC activity. In fact, WNK4 could play a role in coordinating transport via the transcellular (NCC) and paracellular (conductive Cl movement) pathways.
WNK1 did not affect NCC activity directly, but instead altered the ability of WNK4 to suppress NCC activity. This indicates a previously unrecognized interaction between WNK kinases. The effect of WNK1 to inhibit WNK4 is nearly as powerful as the effect of WNK4 to inhibit NCC activity. The effect of WNK1 on WNK4 has the net result of increasing NCC activity. Wilson and colleagues (4
) reported that PHAII-causing WNK1 mutations increase WNK1 expression. An increase in WNK1 expression would suppress WNK4 activity, activating NCC, and could lead to the PHAII phenotype.
The phosphorylation targets of WNK1 are unknown. While WNK1 may phosphorylate WNK4, recent results suggest that the kidney-specific WNK1 lacks a kinase domain. Xu et al. (23
) found that WNK1 contains an autoinhibitory domain between amino acids 510 and 540, centered around two important phenylalanine moieties at 524 and 526. This FXF
motif binds to the catalytic domain and inhibits its activity. Both this autoinhibitory domain and the kinase domain of WNK1 are highly conserved in WNK4, raising the possibility that the autoinhibitory domain from WNK1 interacts with WNK4.
WNK1 is activated by hypertonicity (5
). Such activation, if it occurs in the distal tubule, may explain the effects of luminal NaCl to alter NCC activity acutely. Increases in luminal NaCl concentration along the DCT lead to rapid increases in NCC activity (22
). This occurs despite the fact that the typical luminal concentrations of Na and Cl are well above the Km
for the transport protein (24
). This suggests that luminal ion concentrations may influence NCC activity allosterically. A hypertonicity-induced stimulation of WNK1 activity could mediate such an effect by increasing NCC activity.
In summary, these results indicate that WNK1 and WNK4 interact to regulate NCC activity in the mammalian distal nephron. They identify a previously unrecognized kinase pathway that may play a central role in controlling extracellular fluid volume. Known regulators of NCC activity may interact with this pathway, a pathway that may also participate in osmolality-induced transport regulation. Mutations in WNK kinases cause PHAII. The mutation Q562E is less active than wild type in inhibiting NCC activity. Thus, WNK mutations probably increase NCC activity, thereby contributing to the PHAII phenotype. While PHAII is rare, a genome-wide screen of hypertensive individuals identified a chromosomal region encompassing WNK4 as an important contributor to blood pressure variability (4
). Thus, WNK kinases, and their ability to regulate NCC, may contribute importantly to human blood pressure variation and provide novel targets for pharmaceutical intervention.